In photoreceptor cells, changes in intracellular levels of cGMP link light detection by rhodopsin with membrane hyperpolarization and synaptic signaling [2]. The turnover of cGMP is strictly controlled by the opposing activities of a phosphodiesterase (PDE), which hydrolyzes cGMP, and guanylate cyclase (GC), that synthesizes cGMP. PDE is activated as a result of light detection by rhodopsin, while GC is activated by the low Ca2+ concentration that follows a light detection event. Regulation of the GC activity requires myristoylated Ca2+ binding proteins called Guanylate Cyclase Activating Proteins or GCAPs [2]. These GCAPs undergo Ca2+ induced conformational changes that mediate their ability to inhibit GC at high Ca2+ concentration and activate GC at low Ca2+ concentration [4-6]. Mutations disrupting GC function or the ability of GCAPs to regulate GC result in retinopathies leading to partial or complete blindness [8]. Four point mutations in GCAP1 including a well characterized Y99C mutation, have been shown to cause cone or cone-rod dystrophies in humans [9-12]. Most of these mutations appear to lock GCAP1 in the GC activating conformation losing the ability to regulate GC as a function of Ca2+ concentration. We need to elucidate the conformational changes undergone by wild type and mutant GCAP1 upon Ca2+ binding and release as well as the molecular basis of GCAP1 interaction with GC. These are crucial steps towards understanding the key role of GCAP-GC in phototransduction and their function in the etiology of retinal dystrophies. As preliminary data we have solved the first structure of a myristoylated GCAP (myrGCAP1) with Ca2+ bound. We propose to carry out structural and biochemical experimentation to probe the Ca2+ induced conformational change that allows GCAPs to modulate the activity of GCs in response to Ca2+. Successful completion of the goals outlined in this proposal will break new ground in our understanding of phototransduction regulation. It will also provide a platform to probe the molecular mechanisms underlying the human retinal dystrophies caused by malfunction of GCAP and GC. PUBLIC HEALTH RELEVANCE: Guanylate Cyclase Activating Proteins or GCAPs are Ca2+ binding proteins that regulate the activity of Guanylate Cyclases (GCs). This regulatory activity is essential for normal physiology of photoreceptor cells in the retina and their malfunction leads to retinal disease and partial or total blindness. This proposal is focused on understanding the GCAP regulation of GCs and the molecular mechanisms underlying disease-causing mutation in GCAP1.